Yu-Feng Huang

Parathyroid Hormone Regulates the Expression of Fibroblast Growth Factor-2 mRNA and Fibroblast Growth Factor Receptor mRNA in Osteoblastic Cells

We examined the effect of parathyroid hormone (PTH) on basic fibroblast growth factor‐2 (FGF‐2) and FGF receptor (FGFR) expression in osteoblastic MC3T3‐E1 cells and in neonatal mouse calvariae. Treatment of MC3T3‐E1 cells with PTH(1–34) (10–8M) or forskolin (FSK; 10–5M) transiently increased a 7 kb FGF‐2 transcript with a peak at 2 h. The PTH increase in FGF‐2 mRNA was maintained in the presence of cycloheximide. PTH also increased FGFR‐1 mRNA at 2 h and transiently increased FGFR‐2 mRNA at 1 h. FGFR‐3 and FGFR‐4 mRNA transcripts were not detected in MC3T3‐E1 cells. In cells transiently transfected with an 1800‐bp FGF‐2 promoter‐luciferase reporter, PTH and FSK increased luciferase activity at 2 h and 4 h. Immunohistochemistry showed that PTH and FSK increased FGF‐2 protein labeling in the nuclei of MC3T3‐E1 cells. PTH also increased FGF‐2 mRNA, and FGFR‐1 and FGFR‐2 mRNA levels within 30 minutes in neonatal mouse calvarial organ cultures. We conclude that PTH and cAMP stimulate FGF‐2 mRNA abundance in part through a transcriptional mechanism. PTH also regulated FGFR gene expression. We hypothesize that some effects of PTH on bone remodeling may be mediated by regulation of FGF‐2 and FGFR expression in osteoblastic cells.

Parathyroid hormone inhibits collagen synthesis and the activity of rat Col1a1 transgenes mainly by a cAMP‐mediated pathway in mouse calvariae

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8‐bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin‐6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50–85% in cultured calvariae carrying transgenes having progressive 5′ upstream deletions of promoter DNA down to −1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL‐6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of −1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter. J. Cell. Biochem. 77:149–158, 2000.

Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

Expression of inducible cAMP early repressor is coupled to the cAMP-protein kinase A signaling pathway in osteoblasts

We previously showed that parathyroid hormone (PTH) induces inducible cAMP early repressor (ICER) in osteoblastic cells and mouse calvariae. PTH signaling in osteoblastic cells is transduced by PTH receptor 1, which is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. In the present study, we examined the role of these pathways in mediating PTH-induced ICER mRNA and protein expression in osteoblastic MC3T3-E1 cells. Using RT-PCR, we found that PTH(1-34), forskolin (FSK), and 8-bromo-cAMP (8Br-cAMP) induced ICER expression, while phorbol myristate acetate (PMA), ionomycin, and PTH(3-34) did not. Similar results were found for the induction of ICER protein. PKA inhibition by H89 markedly reduced PTH- and FSK-induced ICER expression, while PKC depletion by PMA had little effect. We also tested ICER induction by other osteotropic signaling agonists. Other cAMP-PKA pathway activators, such as PTH-related protein (PTHrP), induced ICER expression, while agents that signal through other pathways did not. PTHrP maximally induced ICER mRNA at 2-4 h, which then returned to baseline by 10 h. Finally, PTH, FSK, and PTHrP induced ICER in cultured mouse calvariae and osteoblastic ROS 17/2.8, UMR-106, and Pyla cells. We conclude that ICER expression in osteoblasts requires activation of the cAMP-PKA signaling pathway.

Identification of Novel cAMP Responsive Element Modulator (CREM) Isoforms Expressed by Osteoblasts

CREM, the cyclic adenosine monophosphate (cAMP) responsive element modulator, belongs to a multigene family of cAMP-responsive transcription factors. CREM encodes a variety of different isoforms by utilizing four promoters and a complex pattern of alternative messenger ribonucleic acid (mRNA) splicing. We showed previously that parathyroid hormone induces the CREM P2 promoter products known as ICER (inducible cAMP early repressor) in osteoblasts. Herein we report that osteoblasts also express at least 15 CREM transcripts initiated from the P1 promoter, including 7 novel transcripts that result from alternative splicing. It is of interest that we found that CREM-X contains both exon θ1, previously identified only in P3 promoter products, and a new exon termed L, which is located upstream of exon θ1.